Abstract

Caveolae are cholesterol-rich, flask-shaped invaginations of the plasma membrane with many roles in the cell, including the regulation of signal transduction. Caveolin 1 (Cav1) is the major protein responsible for the organization and maintenance of caveolae microdomains. Cav1 recruits many receptor and non-receptor tyrosine kinases and through binding to its scaffolding-domain, Cav1 sequesters the kinases in an inactive form, thereby preventing their involvement in signaling pathways. One important downstream target of many tyrosine kinases (Src, EGF, PDGF, IL6 and others) is the signal transducer and activator of transcription-3 (Stat3). Stat3 is a cytoplasmic signal transducer which is activated by tyrosine-705 phosphorylation by a number of kinases, then migrates to the nucleus to initiate transcription of genes involved in cell division and survival. Despite extensive evidence on the role of cav1 in signal transduction, its effect upon Stat3 is still obscure.

We previously demonstrated that cell-to-cell adhesion, as occurs in confluent cultures, can cause a dramatic increase in Stat3 phosphorylation and activity in cultured cells (Oncogene 23:2600, MBC 16:3832). Therefore, to examine effects upon Stat3, cell density has to be taken into account. Our results now indicate that cav-1 downregulation through expression of an anti-sense construct, or treatment with the pharmacological inhibitor, methyl-cyclo-dextran which removes cholesterol from the membrane and destroys caveolae, caused a strong activation of Stat3 as well as Erk1/2, at all densities examined. Conversely, cav1 overexpression downregulated Stat3 and induced apoptosis in NIH3T3 fibroblasts both before and after transformation by the Simian Virus 40 Large Tumor antigen, as well as in HeLa cells. In all cases, apoptosis was prevented by co-expression of the constitutively active form of Stat3, Stat3C. Taken together, these findings point to cav1 as an inhibitor of Stat3 activity.

It was previously demonstrated that cav1 upregulates p53 gene activity. Since Stat3 is known to inhibit p53 transcription by direct binding to the p53 promotor, these data also point to the possibility that cav1 may, in fact, activate p53 through Stat3 inhibition.

Our results also demonstrate for the first time that, in a feedback loop, Stat3 inhibition following infection with an Adenovirus vector expressing a Stat3-specific, shRNA, results in a dramatic increase in cav1 levels, indicating that Stat3 also downregulates cav1 expression. Since p53 also upregulates cav1, and Stat3 blocks the p53 promotor, it is possible that Stat3 may block cav1 simply by downregulating p53, rather than downregulating the cav1 promotor directly. The above findings taken together reveal the presence of a potent, negative regulatory loop between cav1 and Stat3 activity that plays a crucial role in cellular survival. (supported by CIHR, CBCF-Ontario chapter, US Army breast cancer program NSERC and BCAK).